Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy

A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated ‘invisible’ protein states that exchange with a ‘visible’ ground state on the millisecond time-scale. The utility of the approach is demonstrated with an application to an I58D mutant of the Pfl6 Cro protein that undergoes exchange between the native, folded state and a cold denatured, unfolded conformational ensemble that is populated at a level of 6% at 2.5°C. A wide distribution of amide temperature coefficients is measured for the unfolded state. The distribution is centered about –5.6 ppb/K, consistent with an absence of intra-molecular hydrogen bonds, on average. However, the large range of values (standard deviation of 2.1 ppb/K) strongly supports the notion that the unfolded state of the protein is not a true random coil polypeptide chain.     

The next evolutionary synthesis: from Lamarck and Darwin to genomic variation and systems biology

The evolutionary synthesis, the standard 20^th century view of how evolutionary change occurs, is based on selection, heritable phenotypic variation and a very simple view of genes. It is therefore unable to incorporate two key aspects of modern molecular knowledge: first is the richness of genomic variation, so much more complicated than simple mutation, and second is the opaque relationship between the genotype and its resulting phenotype. Two new and important books shed some light on how we should view evolutionary change now. Evolution: a view from the 21 ^st century by J.A. Shapiro (2011, FT Press Science, New Jersey, USA. pp. 246. $34.99.) examines the richness of genomic variation and its implications. Transformations of Lamarckism: from Subtle Fluids to Molecular Biology edited by S.B. Gissis &; E. Jablonka (2011, MIT Press, Cambridge, USA. pp. 457) includes some 40 papers that anyone with an interest in the history of evolutionary thought and the relationship between the environment and the genome will want to read. This review discusses both books within the context of contemporary evolutionary thinking and points out that neither really comes to terms with today's key systems-biology question: how does mutation-induced variation in a molecular network generate variation in the resulting phenotype?     

Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15–19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.Since its first demonstration in (live) cell imaging (1), stimulated emission depletion (STED) fluorescence microscopy has been realized in many variants. Particularly, the key phenomenon employed in this method, namely switching fluorophores transiently off by stimulated emission, has been accomplished with laser pulses varying from picoseconds to nanoseconds in duration, and from kHz to MHz in repetition rate. Because continuous-wave beams are suitable as well (2), STED microscopy has been implemented with rather different laser systems, ranging from model-locked femtosecond to continuous-wave laser diodes (3,4). Although it underscores the versatility of STED to modulate the fluorescence capability of a fluorophore, this wide range of options may confuse adopters when balancing simplicity, applicability, and resolution gain. The situation is exacerbated when implementing pairs of excitation and STED beams for dual-color colocalization studies (5,6).Here we report on a simple arrangement providing dual-color STED nanoscopy (Fig. 1) and molecular diffusion quantification down to ∼20 nm in (living) cells. The presented dual-channel STED microscope utilizes a single fiber laser providing a 20-MHz train of 775 nm wavelength pulses of 1.2-ns duration. This compact laser source enables STED on fluorophores emitting in the orange to red range. Specifically, we applied this laser on the orange dyes Atto590 and Atto594 (excitation: 595 nm; detection: 620 ± 20 nm), and the red dyes KK114 and Abberior Star635P (excitation: 640 nm; detection: 670 ± 20 nm). Although the spectra of the dyes are partially overlapping, the individual color channels can be separated without data processing (see Fig. S1 and Fig. S2 in the Supporting Material). Both channels are recorded simultaneously within 50 ns, using temporally interleaved pulsed excitation in combination with time-gated detection (5,7,8).Open in a separate windowFigure 1Fluorescence nanoscopy of protein complexes with a compact near-infrared nanosecond-pulsed STED microscope. (A) STED reveals immunolabeled subunits in amphibian NPC; raw data smoothed with a Gaussian filter extending over 14 nm in FWHM. The diameter of the octameric gp210 ring is established as ∼160 nm. Scale bar, 500 nm. (B) Individual NPC image showing eight antibody-labeled gp210 homodimers as 20–40 nm sized units and a 80 nm-sized localization of the subunits in the central channel.Because in STED microscopy, the STED doughnuts firmly determine the position of the fluorescently active molecules, the use of a single doughnut for both fluorophores guarantees that the two color channels are almost perfectly coaligned. The use of the doughnut even counteracts misalignments of the confocal excitation and detection channels (Fig. 2, and see Fig. S3), making STED microscopy particularly powerful for colocalization measurements.Open in a separate windowFigure 2Determination of the colocalization accuracy. Xenopus A6 cells, labeled with an antiserum against multiple NUP subunits in the central NPC channel and two secondary antibodies decorated with the fluorophores Abberior STAR635P and Atto594 were imaged by STED microscopy. (A) Upon overlaying both channels, a high degree of colocalization is directly visible. Scale bar, 200 nm. (B) Quantification of the colocalization by cross correlation of much larger images (see Fig. S3). The correlation is maximal for zero displacement of the images, proving colocalization. (C) Confocal image of monocolored fluorescent beads taken with improperly coaligned excitation beams (left). Improper coalignment spoils the colocalization accuracy in confocal imaging; the two channels should be perfectly coaligned, but they show a false offset as indicated by the color difference. The offset is quantified by the cross correlation of the two channels (right). (D) The STED image of the same beads (left) not only shows 10-fold improved resolution over the confocal image in panel C, but also improved colocalization, again quantified by cross correlation (right). Thus, by predetermining the position of emission, the STED doughnut counteracts errors induced by imperfect coalignment of the two confocal color channels (for details, see Fig. S3). Scale bars = 100 nm.The cross section for stimulated emission is lower at 775 nm as compared to that found at somewhat shorter wavelengths (5), yet STED pulse energies of ∼7 nJ in the focus are sufficient to yield a resolution of ∼30 nm and ∼20 nm in the orange and red channels, respectively (see Fig. S4). In addition, due to the lower peak intensity, the 1.2 ns pulses are likely to induce less nonlinear absorption and hence less photostress as compared to their more commonly used <0.2 ns counterparts (8,9). On the other hand, the pulses are only 2–4 times shorter than the typical lifetime of the excited state, which lessens their STED efficiency. This slight reduction is neutralized here by detecting photons emitted ∼1 ns after excitation (5,7,8).The potential of this straightforward implementation of STED microscopy is evident when imaging immunolabeled nuclear pore complexes (NPCs) of cultured Xenopus cells. Contrary to the confocal recording, STED microscopy reveals subunits of this protein complex, specifically the typical eightfold symmetry of its peripheral transmembrane protein gp210, along with a set of proteins in the central pore channel (Fig. 1, and see Fig. S5 and Fig. S6). Unlike in stochastic superresolution imaging of gp210 (10), the color channels are inherently coaligned and simultaneously recorded simply by executing a single scan. Apart from a weak smoothing and background subtraction applied to enhance image contrast, the images are raw.Because fluorescence off-switching by STED is an instant process, STED microscopy can be employed to study fast spatial translocations, such as the diffusion of molecules on the nanoscale (3). To benchmark the performance of our setup, we analyzed the diffusion of a fluorescent glycerophospholipid analog (11) by fluorescence correlation spectroscopy (FCS) in membranes of living mammalian PtK2-cells (Fig. 3). STED allowed us to reduce the diameter of the probed area from the 250 nm-sized diffraction limit down to 19 nm (FWHM), representing σ = 8 nm in standard deviation of a Gaussian fit. The attained subdiffraction area is 2.5 times smaller as compared to what has been reported in living cells to date (4). In model membranes, the smallest diameter was 15 nm (σ = 6.4 nm).Open in a separate windowFigure 3Nanoscale molecular diffusion analyzed by STED FCS. (A) For moderate and larger STED beam power P_STED, the resolution scales inversely with its square-root, attaining 15 nm in FWHM of the distribution of fluorescence emission in space, describing the measurement area. Note the relatively small threshold power P_S = 1.4 mW, which implies that a large resolution gain is already attained for P_STED < 100 mW. (Inset) The resolution was determined by measuring the transit time of a fluorescent phospholipid-analog (DSPE-PEG-KK114) in a lipid model membrane through the detection area by FCS. (B) In living mammalian Ptk2-cells, the transit time of the lipid analog scales linearly with the detection area, revealing a diffusion constant D_lat = 0.33 μm²/s, and showing that this lipid analog diffuses largely freely in the plasma membrane down to <20 nm scales.In both measurements, the molecular transit time depends linearly on the probed area, indicating that the labeled lipid molecules diffuse essentially freely down to spatial scales of 20 nm. Accordingly, the anomaly exponent α was close to 1 with values of α > 0.85, showing only minor deviations from free diffusion (see Fig. S7). Because the diameter is inversely proportional to the square-root of the STED beam power, the resolution can be adapted to a particular application need (Fig. 3, A and B).In summary, our arrangement provides up-to-date STED microscopy resolution in offset-free colocalization recordings. The ready-to-use near-infrared laser pulses keep undesired single and multiphoton absorption low and leave the visible spectrum amenable for further studies.     

Identification of Protein p270/Tpr as a Constitutive Component of the Nuclear Pore Complex–attached Intranuclear Filaments

Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M_r ~270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515–1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/ Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC- attached filaments and discuss its possible functions.     

Evolutionary dynamics of origin and loss in the deep history of phospholipase D toxin genes

Background

Venom-expressed sphingomyelinase D/phospholipase D (SMase D/PLD) enzymes evolved from the ubiquitous glycerophosphoryl diester phosphodiesterases (GDPD). Expression of GDPD-like SMaseD/PLD toxins in both arachnids and bacteria has inspired consideration of the relative contributions of lateral gene transfer and convergent recruitment in the evolutionary history of this lineage. Previous work recognized two distinct lineages, a SicTox-like (ST-like) clade including the arachnid toxins, and an Actinobacterial-toxin like (AT-like) clade including the bacterial toxins and numerous fungal homologs.

Results

Here we expand taxon sampling by homology detection to discover new GDPD-like SMase D/PLD homologs. The ST-like clade now includes homologs in a wider variety of arthropods along with a sister group in Cnidaria; the AT-like clade now includes additional fungal phyla and proteobacterial homologs; and we report a third clade expressed in diverse aquatic metazoan taxa, a few single-celled eukaryotes, and a few aquatic proteobacteria. GDPD-like SMaseD/PLDs have an ancient presence in chelicerates within the ST-like family and ctenophores within the Aquatic family. A rooted phylogenetic tree shows that the three clades derived from a basal paraphyletic group of proteobacterial GDPD-like SMase D/PLDs, some of which are on mobile genetic elements. GDPD-like SMase D/PLDs share a signature C-terminal motif and a shortened βα1 loop, features that distinguish them from GDPDs. The three major clades also have active site loop signatures that distinguish them from GDPDs and from each other. Analysis of molecular phylogenies with respect to organismal relationships reveals a dynamic evolutionary history including both lateral gene transfer and gene duplication/loss.

Conclusions

The GDPD-like SMaseD/PLD enzymes derive from a single ancient ancestor, likely proteobacterial, and radiated into diverse organismal lineages at least in part through lateral gene transfer.

     

Using Stable Isotopes to Infer the Impacts of Habitat Change on the Diets and Vertical Stratification of Frugivorous Bats in Madagascar

Human-modified habitats are expanding rapidly; many tropical countries have highly fragmented and degraded forests. Preserving biodiversity in these areas involves protecting species–like frugivorous bats–that are important to forest regeneration. Fruit bats provide critical ecosystem services including seed dispersal, but studies of how their diets are affected by habitat change have often been rather localized. This study used stable isotope analyses (δ¹⁵N and δ¹³C measurement) to examine how two fruit bat species in Madagascar, Pteropus rufus (n = 138) and Eidolon dupreanum (n = 52) are impacted by habitat change across a large spatial scale. Limited data for Rousettus madagascariensis are also presented. Our results indicated that the three species had broadly overlapping diets. Differences in diet were nonetheless detectable between P. rufus and E. dupreanum, and these diets shifted when they co-occurred, suggesting resource partitioning across habitats and vertical strata within the canopy to avoid competition. Changes in diet were correlated with a decrease in forest cover, though at a larger spatial scale in P. rufus than in E. dupreanum. These results suggest fruit bat species exhibit differing responses to habitat change, highlight the threats fruit bats face from habitat change, and clarify the spatial scales at which conservation efforts could be implemented.     

Dispersal patterns of oribatid mites across habitats and seasons

Oribatid mites are tiny arthropods that are common in all soils of the world; however, they also occur in microhabitats above the soil such as lichens, mosses, on the bark of trees and in suspended soils. For understanding oribatid mite community structure, it is important to know whether they are dispersal limited. The aim of this study was to investigate the importance of oribatid mite dispersal using Malaise traps to exclude sole passive wind-dispersal. Oribatid mite communities were collected over a 3-year period from five habitat types (coniferous forests, deciduous forests, mixed forests, meadows, bog/heathlands sites) and three seasons (spring, summer, autumn) in Sweden. Mites entered traps either by walking or by phoresy, i.e., by being attached to flying insects. We hypothesized (1) that oribatid mite communities in the traps differ between habitats, indicating habitat-limited dispersal, and (2) that oribatid mite communities differ among seasons suggesting that dispersal varies due to changing environmental conditions such as moisture or resource availability. The majority of the collected species were not typically soil-living species but rather from habitats such as trees, lichens and mosses (e.g., Carabodes labyrinthicus, Cymbaeremaeus cymba, Diapterobates humeralis and Phauloppia lucorum) indicating that walking into the traps or entering them via phoresy are of greater importance for aboveground than for soil-living species. Overall, oribatid mite communities collected in the traps likely originated from the surrounding local habitat suggesting that long distance dispersal of oribatid mites is scarce. Significant differences among seasons indicate higher dispersal during warm and dry periods of the year. Notably, 16 species of oribatid mites collected in our study were sampled for the first time in Sweden. This study also demonstrates that Malaise traps are a meaningful tool to investigate spatial and temporal patterns of oribatid mite communities.

     

Testing the depth-differentiation hypothesis in a deepwater octocoral

The depth-differentiation hypothesis proposes that the bathyal region is a source of genetic diversity and an area where there is a high rate of species formation. Genetic differentiation should thus occur over relatively small vertical distances, particularly along the upper continental slope (200–1000 m) where oceanography varies greatly over small differences in depth. To test whether genetic differentiation within deepwater octocorals is greater over vertical rather than geographical distances, Callogorgia delta was targeted. This species commonly occurs throughout the northern Gulf of Mexico at depths ranging from 400 to 900 m. We found significant genetic differentiation (F_ST = 0.042) across seven sites spanning 400 km of distance and 400 m of depth. A pattern of isolation by depth emerged, but geographical distance between sites may further limit gene flow. Water mass boundaries may serve to isolate populations across depth; however, adaptive divergence with depth is also a possible scenario. Microsatellite markers also revealed significant genetic differentiation (F_ST = 0.434) between C. delta and a closely related species, Callogorgia americana, demonstrating the utility of microsatellites in species delimitation of octocorals. Results provided support for the depth-differentiation hypothesis, strengthening the notion that factors covarying with depth serve as isolation mechanisms in deep-sea populations.     

Trail‐sharing among tropical ants: interspecific use of trail pheromones?

1. Trail‐sharing between different ant species is rare and restricted to a small number of species pairs. Its underlying mechanisms are largely unknown. For trail‐sharing to occur, two factors are required: (i) one or both species must recognise the other species or its pheromone trails and (ii) both species must tolerate each other to a certain extent to allow joint use of the trail. A species that follows another's trails can efficiently exploit the other's information on food sources contained in the pheromone trails. Hence, food competition and thus aggressive interactions between a species following another's trail and the species being followed, seem likely. 2. In the present study, we investigated interspecific trail following and interspecific aggression in trail sharing associations (i) among Polyrhachis ypsilon, Camponotus saundersi, and Dolichoderus cuspidatus, and (ii) among Camponotus rufifemur and Crematogaster modiglianii. We tested whether trail‐sharing species follow each other's pheromone trails, and whether the ants tolerated or attacked their trail‐sharing partners. In both associations, we confronted workers with pheromone trails of their associated species, and, for the former association, measured interspecific aggression among the trail‐sharing species. 3. In our assays, D. cuspidatus and C. rufifemur regularly followed heterospecific pheromone trails of P. ypsilon and C. modiglianii, respectively. However, only few workers of the remaining species followed heterospecific pheromone trails. Thus, shared trails of P. ypsilon and C. saundersi cannot be explained by interspecific trail‐following. 4. Interspecific aggression among P. ypsilon, C. saundersi, and D. cuspidatus was strongly asymmetric, C. saundersi being submissive to the other two. All three species differentiated between heterospecific workers from the same or another site, suggesting habituation to the respective trail‐sharing partners. We therefore hypothesise that differential tolerance by dominant ant species may be mediated by selective habituation towards submissive species and this way determines the assembly of trail‐sharing associations.